Evidence for a conformational change in actin induced by fimbrin (N375) binding.

Hanein D, Matsudaira P, DeRosier DJ - J. Cell Biol. (1997)

Bottom Line:
In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers.The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375.This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself.

ABSTRACTFimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

Figure 5: Schematic diagram of one actin subunit in the F-actin filament (a). The conformational change induced by N375 binding to F-actin is schematically presented in b. Subdomain 1 is shifted outward and downward in a direction away from the bound N375.

Mentions:
Our difference maps show a significant positive peak (2) centered at the COOH terminus of subdomain 1 (Figs. 3 and 4 d). This peak is unlikely to be an artefact resulting from noise in the images or from mishandling of the data for the following reasons: (a) The peak persists whether all the data or just the statistically significant data are included in the difference map. (b) The peak persists when the difference map is calculated in real (density) or reciprocal (Fourier) space. (c) The peak is present when an independent N375 data set is used and when an independent actin data set is used (data not shown). (d) The same peak has been found independently in studies of actin decorated with calponin (Hodgkinson et al., 1997), which has an ABD that is homologous to each of the two CH domains in N375. It is important to emphasize that calponin does not have the 12-kD calcium binding domain of N375 (the headpiece), eliminating the option that peak 2 is caused by the headpiece. Therefore, we must take the peak as a real feature of the difference map, which lies in the midst of the actin portion of the map. The variance in this region in both maps is low, suggesting that the shift is not the result of a change in the mobility of the domain. There are also two statistically significant negative regions in the difference map. One of them is centered near the NH2 terminus of subdomain 1, and the other is located near subdomain 2. This information suggests that subdomain 1 is shifted outward and downward in a direction away from the bound N375 subunit (see Fig. 5).

Figure 5: Schematic diagram of one actin subunit in the F-actin filament (a). The conformational change induced by N375 binding to F-actin is schematically presented in b. Subdomain 1 is shifted outward and downward in a direction away from the bound N375.

Mentions:
Our difference maps show a significant positive peak (2) centered at the COOH terminus of subdomain 1 (Figs. 3 and 4 d). This peak is unlikely to be an artefact resulting from noise in the images or from mishandling of the data for the following reasons: (a) The peak persists whether all the data or just the statistically significant data are included in the difference map. (b) The peak persists when the difference map is calculated in real (density) or reciprocal (Fourier) space. (c) The peak is present when an independent N375 data set is used and when an independent actin data set is used (data not shown). (d) The same peak has been found independently in studies of actin decorated with calponin (Hodgkinson et al., 1997), which has an ABD that is homologous to each of the two CH domains in N375. It is important to emphasize that calponin does not have the 12-kD calcium binding domain of N375 (the headpiece), eliminating the option that peak 2 is caused by the headpiece. Therefore, we must take the peak as a real feature of the difference map, which lies in the midst of the actin portion of the map. The variance in this region in both maps is low, suggesting that the shift is not the result of a change in the mobility of the domain. There are also two statistically significant negative regions in the difference map. One of them is centered near the NH2 terminus of subdomain 1, and the other is located near subdomain 2. This information suggests that subdomain 1 is shifted outward and downward in a direction away from the bound N375 subunit (see Fig. 5).

Bottom Line:
In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers.The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375.This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself.

ABSTRACTFimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.